Chlorine gas is a highly toxic respiratory irritant that is considered a chemical threat agent. The goal of the research proposed in this renewal application is to develop a medical countermeasure to treat lung injury induced by chlorine inhalation. During the previous U01 award, a mouse model of chlorine gas inhalation was developed, a detailed characterization of lung injury following chlorine exposure was performed, signaling pathways representing possible targets for therapeutic intervention were investigated, strategies for manipulating signaling pathways of interest were tested, and compounds that significantly inhibited lung injury or inflammation in chlorine-exposed mice were identified. Rolipram, a type 4 phosphodiesterase inhibitor, was shown to stimulate alveolar fluid transport, decrease pulmonary edema, and inhibit airway hyperreactivity. Triptolide, a diterpenoid anti-inflammatory compound, inhibited chlorine-induced lung inflammation. In the present application, formulations of rolipram and triptolide will be developed for intramuscular (i.m.) injection, a preferred delivery route for potential mass casualty situations. In addition, methylprednisolone, an FDA-approved anti-inflammatory compound, will be evaluated as an alternative to triptolide. The overall hypothesis to be tested is that i.m. injection of an encapsulated formulation of rolipram in combination with an anti-inflammatory compound will represent the most effective countermeasure for chlorine-induced lung injury. Countermeasure formulations will be tested in mice to determine efficacy against chlorine-induced acute lung injury, to assess longer term effects on airway remodeling and airway hyperreactivity, and to measure the length of effective post-exposure treatment window. The most effective countermeasure formulation will be evaluated in rabbits to demonstrate efficacy in a non-rodent animal model. An optimized countermeasure formulation with efficacy against chlorine-induced lung injury in mice and rabbits will be subject to additional studies necessary for progression toward regulatory approval, including collection of pharmacokinetic/pharmacodynamic and toxicity data and production of the countermeasure using current good manufacturing practices. Public Health Relevance: The goal of the proposed experiments is to develop novel ways to treat acute lung injury caused by chlorine gas inhalation. The research will focus on medical countermeasures that can be easily and rapidly administered in a mass casualty situation. This type of research is important because of concerns that U.S. civilians could be adversely affected by the accidental or intentional release of highly toxic chemicals.